AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 281: H1223-H1232, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (16)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhai, P.
Right arrow Articles by Gross, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhai, P.
Right arrow Articles by Gross, D. R.
Vol. 281, Issue 3, H1223-H1232, September 2001

Effects of dietary phytoestrogen on global myocardial ischemia-reperfusion injury in isolated female rat hearts

Peiyong Zhai1, Thomas E. Eurell2, Robert P. Cotthaus2, Elizabeth H. Jeffery3, Janice M. Bahr4, and David R. Gross2

1 Division of Cardiology, Department of Medicine, Johns Hopkins Medical Center, Baltimore, Maryland 21224; and 2 Department of Veterinary Biosciences, 3 Department of Food Science and Human Nutrition, and 4 Department of Animal Sciences, University of Illinois, Urbana-Champaign, Illinois 61802


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effects of phytoestrogen on global myocardial ischemia-reperfusion injury in five groups of female rats. A high-phytoestrogen group (HPE) was ovariectomized (Ovx) and fed a diet containing soybean protein and a high-isoflavone soy extract. Another Ovx group of rats was fed the same diet as the HPE group but treated with the estrogen receptor blocker ICI-182,780 (HPE + ICI). A third group of Ovx rats was fed a diet containing soybean protein alone (low-phytoestrogen content; LPE). A fourth Ovx group was fed a diet free of phytoestrogen (Ovx). The fifth group of rats was sham ovariectomized (sham). Hearts from all rats were subjected to 30 min of global, hypothermic (4°C), cardioplegic ischemia and 120 min of normothermic (37°C) reperfusion with oxygenated Krebs-Henseleit buffer. Compared with either the sham or the HPE group, the Ovx and HPE + ICI groups had significantly decreased first derivative of left ventricular pressure (dP/dt), coronary flow rate (CFR), nitrite production and mitochondrial respiratory function and significantly increased Ca2+ accumulation and myocardial histological and ultrastructural injury. The CFR of the LPE group was significantly different from that of either Ovx or HPE + ICI group but the dP/dt, nitrite production, Ca2+ accumulation, and mitochondrial function were not. Our results indicate that diets containing phytoestrogen extract play a cardioprotective role in global myocardial ischemia-reperfusion in female rats.

calcium; nitric oxide; cardioplegia; mitochondrial function; myocardial ultrastructure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WE HAVE PREVIOUSLY REPORTED that estrogen has protective effects against myocardial injury caused by global, hypothermic, cardioplegia-induced myocardial ischemia, followed by crystalloid reperfusion in both male mice (37) and female rats (38). Estrogen has also been reported to inhibit high-potassium cardioplegia-induced Ca2+ overload in cultured cardiac myocytes (17).

Phytoestrogens are natural nonsteroidal plant-derived compounds. The major classes of phytoestrogens of current interest are isoflavones. Soy products are rich sources for isoflavones. The primary soy-derived isoflavones are genistein, daidzein, and glycitein. Their structures are similar to the structure of estrogen, and they bind to estrogen receptors (20).

A diet containing soy proteins was found to lower plasma concentration of total cholesterol, low-density lipoprotein cholesterol, and very-low-density cholesterol and increase plasma concentrations of high-density lipoprotein cholesterol in young women (36). Female macaques fed a soy protein diet for 6 mo demonstrated enhanced coronary dilatation in response to acetylcholine. Those fed a similar soy protein diet, but with isoflavones removed by ethanol extraction, demonstrated constriction in response to acetylcholine (14). Genistein and daidzein inhibited mitogen-induced proliferation, migration, and extracellular matrix synthesis in cultured human aortic smooth muscle cells (7). Genistein was shown to inhibit intimal thickening and replication in carotid arteries after denudation injury in rats in vivo and to inhibit smooth muscle cell proliferation and migration in vitro (24). Intravenous administration of 1 mg/kg genistein to rats 5 min after the occlusion of the left main coronary artery lowered myocardial necrosis, decreased serum creatine kinase activity, increased myocardial contractility, and decreased the occurrence of ventricular arrhythmias (6). It is unknown whether dietary phytoestrogen has any effect on myocardial ischemia-reperfusion injury.

The experiments reported here were designed to 1) demonstrate if dietary phytoestrogen has any protective activity in global, cardioplegia-protected ischemia, followed by reperfusion, and, if so, 2) to gain information about the potential mechanisms for the cardioprotective effects.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign and were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Fifty female Sprague-Dawley rats (3 mo old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and fed standard rat chow for 3 mo. At an age of 6 mo, 10 rats were prepared as sham-operated controls (sham). All other rats (n = 40) were ovariectomized (Ovx) and divided into four groups. The anesthesia, Ovx, and sham surgery procedures have been previously described (38). Briefly, the rats were anesthetized with ketamine (20 µg/g ip) and xylazine (0.5 µg/g ip). On each side, the skin was prepared for aseptic surgery and a lateral paralumbar incision was made. The ovary on that side was isolated and removed with the oviduct. Sham animals had the ovaries isolated but left intact. The incisions were closed with stainless steel wound clips that were removed in 7-10 days. Each group was fed one of the diets described in Table 1. The phytoestrogen isoflavone content in the diet containing soybean protein and soybean protein plus soy extract is summarized in Table 2. The Ovx control rats (n = 10) and sham rats were fed a diet supplemented with 200 g/kg of casein, i.e., no dietary phytoestrogen. The low-phytoestrogen (LPE) diet group (n = 10) received 200 g/kg soybean proteins instead of the casein, and the high-phytoestrogen diet groups [HPE, n = 10, and HPE + ICI-187,780 (ICI), n = 10] were both fed 200 g/kg soy protein diets plus 34.4 g/kg high-isoflavone soy protein extract. The HPE + ICI group was additionally treated with a weekly subcutaneous injection of ICI in castor oil vehicle at a dose of 10 mg/kg. The dose of ICI was selected based on results of a study by other faculty in the same department. This study (26) showed maximum testicular effects in rats from the same dose. Rats were fed the special diets for 3 mo and used in the described experimental protocol when they were approaching reproductive senescence, i.e., >9 mo old (1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Diet composition list of the phytoestrogen-free (control) diet, LPE diet, and HPE diet


                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Content of isoflavones in the LPE diet and HPE diet

Experimental protocol. Rats were anesthetized as previously described and treated with heparin (1,000 IU ip). The heart was quickly removed, weighed, and immersed in cardioplegic solution [Plegisol (Abbott Labs) plus 25 mmol/l NaHCO3 and 2 U/ml heparin]; pH 7.4 at 4°C. The same cardioplegic solution was infused retrograde into the coronary arteries through an aortic catheter with the use of a speed-controlled roller pump at an infusion rate of 0.3 ml/min for 5 min. A balloon-tipped catheter was inserted into the left ventricle (LV) via the left atrium and secured. Infusion of the cardioplegic solution was then stopped, and the heart was immersed in the same cold cardioplegic solution for a total ischemia time of 30 min. The heart was mounted in a Langendorff-type isolated heart perfusion system with a water jacket-warmed organ chamber, oxygenator, and reservoir. The hearts were subjected to 2 h of retrograde coronary artery reperfusion with Krebs-Henseleit bicarbonate buffer (Sigma) oxygenated with 95% O2-5% CO2, pH 7.4 and 37°C, at a constant pressure of 120 cmH2O. All hearts were converted to sinus rhythm spontaneously and were allowed to beat at their own rhythm, i.e., not paced. LV pressure (LVP) was continuously measured for the duration of reperfusion using a Digi-MED Heart Performance Analyzer-tau (Micro-Med). Heart rate, maximum LVP, end-diastolic LVP, and the first derivative of LVP (dP/dt) were continuously recorded using a computer installed with Digi-MED System software (MicroMed). The end-diastolic LVP was set so as not to exceed 10 mmHg by adjusting the volume in the balloon at the beginning of the perfusion period. The balloon volume was then kept constant throughout the 120-min reperfusion time. Coronary flow rate (CFR), coronary nitrite concentration, and Ca2+ concentrations in both coronary inflow and effluent were measured during the 120 min of reperfusion as previously described (36). Briefly, coronary flow rate (in ml · min-1 · 100 g-1) was measured by collecting the coronary effluent volume for a total of 120 min. This volume was divided by the time and normalized by the wet weight of the heart (g), measured at the beginning of the experiment. Nitrite production (nmol/g) was estimated as the product of coronary nitrite concentration, which was measured using the Griess reaction (12), and coronary effluent volume normalized by heart wet weight (g), which was not significantly different among the experimental groups. Myocardial Ca2+ accumulation (µmol/g) was estimated from the difference in Ca2+ concentration (µmol/ml) and was measured using inductively coupled plasma atomic emission spectrometry between perfusate and effluent. This was normalized by coronary effluent volume (ml) and the heart wet weight (g).

After 120 min of reperfusion, mitochondrial function was estimated by the conversion of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) to an insoluble formazan dye (20). A 1-mm-thick section of right ventricle and LV was incubated with 1 ml of Dulbecco's modified Eagle's medium without phenol red plus 1 ml of 0.5 mg/ml MTT solution for 24 h at 37°C. The MTT media solution was then gently aspirated, and the formazan dye was extracted from the tissue with 0.5 ml of isopropanol and 0.5 ml of dimethyl sulfoxide. The absorbance at 570 nm was measured and corrected for tissue wet weight (g).

Myocardial histomorphology was evaluated using hematoxylin-eosin (H-E)- and hematoxylin-basic fuchsin-picric acid (HBFP)-stained tissue sections. Four digital images of each sample were randomly taken for morphometric analysis using NIH Image software. The contrast, threshold, and magnification of all images were identical. The percentage of myocardium with a positive HBFP stain was calculated. The percentage of interstitial space in the myocardial tissue was determined on H-E-stained sections. The myocardial ultrastructure was studied using ultrathin (0.1 µm) sections, which were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate, and examined using a Hitachi 600 transmission electron microscope. Four negative films per sample were randomly taken whenever possible from areas where longitudinal myofibrils were observed so that the influence of mitochondrial orientation on mitochondrial size could be minimized. The films were then scanned to obtain digital images, which were analyzed using NIH Image software. The mitochondrial cross-sectional area was measured. The number of fragmented mitochondria, the number of mitochondria with amorphous matrix densities or granular densities, and the total number of mitochondria studied in each group were counted.

Measurement of plasma isoflavones. At the time the heart was isolated, blood plasma was collected and stored at -20°C until used for measuring isoflavones. The concentration of genistein and daidzein in plasma were measured using a high-performance liquid chromatograph and mass spectrometry (HPLC-MS) method (4). The isoflavone conjugates in plasma were hydrolyzed by using beta -glucuronidase-sulfatase (Helix pomatia type H-2, Sigma). A 0.5-ml sample of plasma was incubated with 609.5 units of beta -glucuronidase and 25 units of sulfatase and 200 µl of 10 nM ammonium acetate buffer at 37°C for 16 h. The isoflavone extracts were then extracted according to the method reported by Lundh et al. (22). The overall recovery efficiency was >80% for both genistein and daidzein (92% for enzyme digestion and 90% for extraction). Biochanin A was used as the internal standard in this assay procedure. The isoflavone extracts were redissolved in 30% acetonitrile in 10 mM ammonium acetate buffer and measured using a Waters 2690 HPLC-MS. HPLC separation was performed on a Waters Xterra MS C18 reverse-phase HPLC column with 3.5-µm particle size and 125-Å pore size under isocratic conditions (40% acetonitrile in 10 mM ammonium acetate) at a flow rate of 0.3 ml/min. The single-ion resolution scan function was used for MS analysis after electrospray-negative ionization. The measuring mass charge ratio was set at 269.2 (genistein) and 253.2 (daidzein), respectively. The peak of interest on the chromatogram was identified according to the chromatogram of genistein and daidzein standards, respectively. The integration of peak areas was carried out using the MassLynx software, provided by the mass spectrometer manufacturer. The peak areas were compared with a series of known concentrations of genistein and daidzein standards to estimate the plasma genistein and daidzein concentrations.

Measurement of plasma estradiol concentration. Plasma (1 ml) was used for measuring 17beta -estradiol concentration using a radioimmunoassay kit, as previously described (38).

Statistical analysis. All data are presented as means ± SE and were first analyzed using a two-way analysis of variance (ANOVA) for repeated measures or a single-factor ANOVA as appropriate. If significant differences were observed, a Bonferroni t-test was applied to compare differences between groups. Statistical analyses were done by running appropriate SAS procedures (SAS Institute; Cary, NC). All proportions were compared using a chi 2 test. The alpha -level was set at 0.05 and adjustment was made to control experiment type I error where appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The body weights of the animals were (in g) sham, 322.5 ± 37.2; Ovx, 378.4 ± 25.8; LPE, 334.3 ± 30.7; HPE, 361.8 ± 24.3; and HPE + ICI, 367.0 ± 21.6. The wet heart weights before reperfusion were (in g) sham, 1.63 ± 0.15; Ovx, 1.58 ± 0.15; LPE, 1.31 ± 0.9; HPE, 1.49 ± 0.13; and HPE + ICI, 1.52 ± 0.16. The heart weight-to-body weight ratios were (in %) sham, 0.51 ± 0.07; Ovx, 0.42 ± 0.05; LPE, 0.39 ± 0.03; HPE, 0.41 ± 0.13; and HPE + ICI, 0.415 ± 0.05. A representative number of hearts from each group were also weighed after reperfusion but in no instance was the weight after reperfusion >0.1-0.02 g than the prereperfusion weight.

Plasma genistein and daidzein concentration. Plasma genistein concentration was 226.02 ± 79.67 ng/ml in those rats fed the HPE diet (HPE and HPE + ICI groups). The average plasma genistein concentration was 20.71 ± 2.33 ng/ml in the LPE group, which was significantly lower than that of the HPE and the HPE + ICI groups (P < 0.01). The concentration of plasma genistein averaged 5.17 ± 0.51 ng/ml in the phytoestrogen-free diet rats (sham and Ovx groups), which was significantly lower than that in the HPE and the HPE + ICI groups (P < 0.0001) and that of the LPE group (P < 0.01). The plasma daidzein concentration was 147.85 ± 50.8 ng/ml in the HPE and the HPE + ICI groups, which was significantly higher than that of the LPE group (18.98 ± 1.9 ng/ml, P < 0.001) and than that of the sham and the Ovx groups (8.1 ± 0.98 ng/ml, P < 0.01).

Plasma 17beta -estradiol concentration. Plasma 17beta -estradiol concentration averaged 60 ± 6 pg/ml in the sham group. The average plasma estradiol concentration of Ovx, HPE, LPE, and HPE + ICI groups was 27 ± 1 pg/ml, which was significantly lower than that of the sham group (P < 0.0001).

LV dP/dt. The average LV dP/dt of the sham group during the 120 min of recording was 2,658.9 ± 92.6 mmHg/s, which was significantly higher than that of the Ovx group (1,756.3 ± 129.2 mmHg/s). HPE significantly improved LV function with an average LV dP/dt of 2,724.6 ± 118.2 mmHg/s, which was significantly higher than that of the Ovx group but not significantly different than that of the sham group (Fig. 1). The estrogen receptor blocker ICI abolished the effect of HPE on LV dP/dt (1,873.4 ± 148.9), significantly lower than that of the HPE group and the sham group but not significantly different from that of the Ovx group (Fig. 1). The LPE group LV dP/dt was not significantly different than that of either the Ovx group or the HPE + ICI group (Fig. 1).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 1.   First derivative of left ventricular (LV) pressure (dP/dt) (mmHg/s, means ± SE). aSignificantly different from the ovariectomized (Ovx) group. bSignificantly different from the high-phytoestrogen (HPE) and ICI-182,780 (ICI)-treated (HPE + ICI) group. Sham, sham-operated group; LPE, low-phytoestrogen-treated group.

Coronary flow rate. The CFR in the sham group averaged 9.07 ± 0.85 ml · min-1 · 100 g-1. The HPE group had a CFR of 10.85 ± 0.48 ml · min-1 · 100 g-1 and the LPE group averaged slightly less. There were no statistically significant differences between the sham, HPE, and LPE groups, but all three had significantly higher flows than the HPE + ICI group (8.12 ± 0.69 ml · min-1 · 100 g-1) and the Ovx group (6.98 ± 0.70 ml · min-1 · 100 g-1) (Fig. 2).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 2.   Coronary flow rate (ml · min-1 · 100 g-1, means ± SE). aSignificantly different from the Ovx group. bSignificantly different from the HPE + ICI group.

Nitrite production. The sham group averaged 2.77 ± 0.44 nmol · min-1 · g-1 nitrite during reperfusion and the HPE group averaged 2.21 ± 0.16 nmol · min-1 · g-1 (Fig. 3). The nitrite production of the Ovx group was 1.05 ± 0.11 nmol · min-1 · g-1, which was significantly less than that of the sham and HPE groups. The HPE + ICI group only produced an average 0.84 ± 0.04 nmol · min-1 · g-1, which was significantly less than that of either the sham group or the HPE group but not the Ovx group. The LPE group was not different than either the Ovx or the HPE + ICI group.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3.   Coronary nitrite production (nmol · min-1 · 100 g-1, means ± SE). aSignificantly different from the Ovx group. bSignificantly different from the HPE + ICI group.

Myocardial Ca2+ accumulation. Over the 120 min of reperfusion, there were no significant differences in the amount of Ca2+ between the perfusate and the effluent in the sham and HPE + ICI groups. The HPE group demonstrated an apparent Ca2+ export, i.e., negative accumulation. There was a significant accumulation of Ca2+ in the hearts of the Ovx and LPE groups compared with the other three groups (Fig. 4).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   Myocardial Ca2+ accumulation (µmol · min-1 · 100 g-1, means ± SE). aSignificantly different from the Ovx group.

Myocardial MTT extraction. After ischemia-reperfusion, myocardial MTT reduction in the Ovx, HPE + ICI, and LPE groups was significantly lower than that in the sham and HPE groups. The latter were not significantly different from each other (Fig. 5). The HPE + ICI group had the lowest myocardial MTT reduction but was not significantly lower than that in the Ovx and LPE groups.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5.   Myocardial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction [optical density (OD)/g, means ± SE]. aSignificantly different from the Ovx group. bSignificantly different from the HPE + ICI group.

Myocardial histology. Representative slides from animals from each group are shown in Figs. 6 and 7. After ischemia-reperfusion, prominent interstitial edema was present on H-E-stained tissue sections from the Ovx (Fig. 6B), the HPE + ICI (Fig. 6D), and the LPE (Fig. 6E) samples, whereas only slight edema was demonstrated in the sham (Fig. 6A) and the HPE samples (Fig. 6C). The amount of edema in the HPE + ICI samples was not different from that of the Ovx samples, and the HPE group was not different from the sham group. These results were obtained from samples from all animals using NIH Image software (Fig. 6F). In the Ovx, the HPE + ICI, and the LPE samples nonuniformly stained myofibrils are visible. This may be the result of loss of contractile materials.


View larger version (117K):
[in this window]
[in a new window]
  		Fig. 6.   Hematoxylin-eosin-stained myocardial samples and results of image analysis. Scale bar, 50 µm. A: sham; myofibrils are shown to be normal. B: Ovx; marked interstitial edema and myofibrils with areas of loss of structures are demonstrated. C: HPE; mild interstitial edema and mild abnormal of myofibrils are demonstrated. D: HPE + ICI; marked interstitial edema and loss of structures within myofibrils are shown. E: LPE; interstitial edema and myofibrils with areas of loss of structure are clearly demonstrated. F: percentage of interstitial space (%, means ± SE). aSignificantly different from the Ovx group. cSignificantly different from the HPE + ICI group.



View larger version (129K):
[in this window]
[in a new window]
  		Fig. 7.   Hematoxylin, basic fuchsin, and picric acid-stained myocardial samples and results of image analysis. Scale bar, 50 µm. A: sham; most of myocytes are not stained (viable) and only a few were positively stained (intensive dark areas), i.e., injured. B: Ovx; most of the myocytes are injured, the extent of which is much more than that of the sham group. C: HPE; few myocytes are injured, the extent of which are not different from that of the sham group but much smaller than that of the Ovx group. D: HPE + ICI; most of myocytes are injured to an extent not different from that of the Ovx group but much more than those of the sham and the HPE groups; interstitial edema is clearly shown. E: LPE; most of myocytes are injured, the extent of which is not different than those of the Ovx and the HPE + ICI groups. F: the percentage of positively stained myocardium (%, means ± SE). aSignificantly different from the Ovx group. cSignificantly different from the HPE + ICI and LPE groups.

Injured myocytes, detected by a positive HBFP stain, were found in all groups, but the numbers of injured cells (those that did not exclude the stain) were more prominent in the Ovx (Fig. 7B) and the HPE + ICI (Fig. 7D) samples than that in the sham (Fig. 7A) or the HPE (Fig. 7C) samples. The LPE samples (Fig. 7E) also demonstrated more severe damage compared with the sham or the HPE samples. The HEP + ICI samples were not different from the Ovx samples, and the HPE samples were not different from the sham samples. These histological impressions were verified quantitatively by the results of the image analysis (Fig. 7F) done on slides from all animals.

Myocardial ultrastructure. Representative transmission electron microscopic results from each group of rats are shown in Fig. 8. The myofibrils were intact in the sham samples (Fig. 8A). The mitochondria were abnormal in appearance, but most of them contained well-defined cristae. No significant intracellular edema was seen. In the Ovx group (Fig. 8B), the clear sarcoplasmic spaces probably indicate intracellular edema and/or loss of normal structures. The mitochondria are obviously abnormal, swollen, and abnormal in shape, and none contain clearly defined cristae. Most demonstrate amorphous matrix densities or granular densities, which may represent aggregation of proteins (such as denature enzymes) and/or deposition of Ca2+ and phosphate (15, 19, 33). Some mitochondria are fragmented and others have their matrix cleared out, resulting in what appears to be vacuoles. The HPE samples (Fig. 8C) are not obviously different from the sham samples, i.e., they show mildly abnormal mitochondria and intact myofibrils. The HPE + ICI samples (Fig. 8D) demonstrate intracellular edema, swollen, fragmented, and vacuolated mitochondria, and mitochondria with amorphous matrix inclusions and/or granular inclusions. Some mitochondria are without clearly defined cristae. These changes are very similar to those seen in the Ovx samples. The LPE samples (Fig. 8E) also showed discontinuation of myofibrils, mild intracellular edema, vacuolated mitochondria, and swollen mitochondria with amorphous matrix inclusions and/or granular inclusions. Most of the mitochondria in these samples did not contain sharply defined cristae. The observed changes in the mitochondria were quantitatively indicated by the results of image analysis conducted on samples from all animals (Fig. 9). The cross-sectional areas of the mitochondria of the sham and HPE groups were the same, but both were significantly smaller than those of the Ovx and HPE + ICI groups (Fig. 9A). There was no significant difference in cross-sectional area between the LPE group and any of the other four. The percentage of mitochondria with granules in the sham and HPE groups was significantly lower than that of the Ovx, HPE + ICI, and LPE groups (Fig. 9B). The percentage of fragmented mitochondria in the sham, HPE and LPE groups was significantly lower than that of the Ovx and the HPE + ICI groups.


View larger version (122K):
[in this window]
[in a new window]
  		Fig. 8.   Transmission electron microscopy of myocardium. Scale bar, 1 µm. A: sham; myofibrils are shown to be intact. Mitochondria are mildly abnormal due to ischemia-reperfusion injury but most contain clearly defined cristae. B: Ovx; mitochondria are prominently abnormal in shape, with abnormal cristae, areas of loss matrix, and amorphous matrix densities and granular densities. Mitochondrial membranes are occasionally disrupted. Clarity of sarcoplasmic space indicates intracellular edema and loss of normal structures. C: HPE; the myofibrils are intact. Mitochondria are slightly abnormal in shape but most contain normal cristae. Occasional and focal loss of matrix is seen in a few mitochondria. D: HPE + ICI; a giant mitochondrion with granular densities and amorphous matrix densities and clarified matrix spaces is shown. One mitochondrion becomes a vacuole. E: LPE; myofibrils are not continued in some. Mild intracellular edema is shown. Mitochondria are abnormal in shape with granular densities and amorphous matrix densities. Some vacuolated mitochondria are shown. Most mitochondria do not contain clearly defined cristae.



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 9.   Results of ultrastructural image analysis. A: mitochondrial cross-sectional area (µm2, means ± SE). B: percentage of mitochondria with amorphous matrix densities or granular densities (%, means ± SE). C: percentage of mitochondria fragmented (%, means ± SE). aSignificantly different from the Ovx group. cSignificantly different from the HPE + ICI group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that dietary phytoestrogen increases circulating genistein and daidzein. Increased circulating genistein and daidzein apparently preserve myocardial structure and improve myocardial function after 30 min of global, hypothermic ischemia, followed by 120 min of normothermic crystalloid reperfusion in mature female rats. Our results also indicate that the protective effects of phytoestrogen in the HPE group are not significantly different from those of endogenous estrogen in the sham-operated group. These results point to a cardioprotective effect of the phytoestrogens genistein and daidzein. The experimental findings in this study do not completely rule out the potential beneficial systemic effects (i.e., on plasma lipids) of dietary phytoestrogens. However, the rat is a poor model of diet-induced cardiovascular disease, so it is difficult to comment on these issues.

Body weights, heart wet weights before reperfusion, and heart weight-to-body weight ratios tended to be less in the LPE group but were not statistically different between the five groups. This result was expected because all groups of rats were fed diets with balanced total caloric intake in controlled amounts.

Only some of the protective effects of phytoestrogens were blocked by treatment with ICI, an anti-estrogen that is reported to block both the alpha - and beta -estrogen receptors. Our results indicated that the protective effects of phytoestrogen were mediated, in part, by interacting with estrogen receptors. Soybean isoflavones are reported to have weak estrogenic activity (5). They bind to both estrogen receptor-alpha and estrogen receptor-beta (20). They reportedly stimulated the transcriptional activity of estrogen receptor that was inhibited by ICI in cell culture experiments (25). After oral intake, genistein labeled with 14C or its metabolites, was shown to accumulate in both reproductive and peripheral tissues where estrogen receptors were present (3). Both estrogen receptors have been demonstrated in the coronary vasculature (30) and the myocardium (12). We (37) previously reported that male estrogen receptor-alpha knockout mice developed ventricular arrhythmias and demonstrated marked myocardial damage following global ischemia-reperfusion, suggesting an important role for estrogen receptor-alpha during ischemia-reperfusion.

One potential mechanism of the cardioprotective effects of phytoestrogens is to maintain nitric oxide (NO) production during ischemia-reperfusion. The basal release of NO from isolated working rat hearts was reduced by ischemia-reperfusion (8). Endothelial NO synthase activity was decreased during ischemia and only partially restored during reperfusion (10). Inhibition of NO synthesis resulted in impaired postischemic recovery of function in isolated hearts (27). NO probably protects the myocardium by improving coronary flow (27) and decreasing the area of no reflow (29) under these experimental conditions. This study indicates that hearts from Ovx rats that had been supplemented with phytoestrogen produced significantly more nitrite than hearts from Ovx rats. This suggests that phytoestrogens maintained NO release. In this study, the effect of phytoestrogen on preserving myocardial systolic function, represented by increased dP/dtmax, did not totally depend on the effect of phytoestrogen on improving coronary flow, i.e., myocardial perfusion, suggesting that flow-mediated mechanisms alone are not sufficient to provide the protection observed. The differences in nitrite production between the LPE group and the Ovx group were not paralleled by those of coronary flow rate in this study, further suggesting that NO may exert protective effects through mechanisms beyond improving coronary flow.

Treatment with ICI in these studies blocked the effect of the phytoestrogen extract on NO release. This suggests phytoestrogens act by interacting with estrogen receptors in maintaining normal NO levels. This may be important because an estrogen response element and activator protein 1, both estrogen receptor-binding motifs, are present in the NO synthase gene (28). In a previous study, subcutaneous administration of genistein at a daily dose of 0.2 mg/kg to Ovx rats for 4 wk increased the activity of Ca2+-dependent NO synthase to the same extent as 17beta -estradiol (34). Both endothelial NO synthase and inducible NO synthase were expressed in endothelial cells, vascular smooth muscle cells, and cardiac myocytes (28). Phytoestrogens may maintain NO production by enhancing the activity and the expression of NO synthase by interacting with estrogen receptors. This, however, does not rule out other possible mechanisms through which phytoestrogens may also regulate NO production.

Preserving mitochondrial structure and function may be another mechanism of cardioprotection by phytoestrogens. It was reported (16) that mitochondria isolated from dog myocardium after 60 min of ischemia had only 3-4% of the capacity to completely oxidize pyruvate to carbon dioxide and water. The activity of electron transfer complex I (NADH-Q reductase) was decreased in ischemic cardiac muscle (32). Structural changes observed in dysfunctional mitochondria from the Jennings et al. (16) study included relatively clear matrices, rare and loosely packed cristae, and dense mitochondrial granules. A reduction in energy production by mitochondria was shown to result in decreased recovery of cardiac function after reperfusion (31). 17beta -Estradiol reportedly stabilized mitochondrial function, i.e., prevented decreases in transmembrane potential and energy charge-redox state, in neurons exposed to apoptotic agents (24). Zheng and Ramirez (39) have demonstrated that the oligomycin-sensitive-conferring protein, a subunit of the F0F1 mitochondrial ATP synthase-ATPase, is required for coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane. The protein plays a role in ATP synthesis in the F1 sector of the enzyme and binds to 17beta -estradiol. The F0F1 mitochondrial ATP synthase-ATPase system is apparently responsible for maintaining Ca2+ homeostasis in the mitochondria. Loss of estrogen may block this function and result in Ca2+ accumulation within the mitochondria. In our previous study (38), estrogen was shown to improve myocardial function by preserving mitochondrial structure and mitochondrial function in female rat hearts. In the present study, dietary phytoestrogen significantly inhibited mitochondrial swelling, reduced the number of mitochondria that were either fragmented, fragmented with granules, or unfragmented with granules, and improved myocardial MTT reduction. Treatment with ICI significantly attenuated the effect of phytoestrogens on mitochondrial function. This suggests that estrogen receptors are involved.

Phytoestrogens may also protect the myocardium against ischemia-reperfusion injury by inhibiting Ca2+ overload during ischemia-reperfusion. The occurrence of Ca2+ overload has been observed in isolated globally ischemic-reperfused rat hearts (35). A large increase in Ca2+ content occurred in severely damaged myocardium during reperfusion after prolonged ischemia (11). Ca2+ overload was reported (2) to depress the recovery of mechanical function of the myocardium. Dietary soy protein extract significantly inhibited Ca2+ accumulation during ischemia-reperfusion in these studies, whereas treatment with ICI did not block this effect. This suggests that phytoestrogens may inhibit Ca2+ entry through mechanisms independent of classical estrogen receptors, possibly related to the oligomycin-sensitive-conferring protein mechanism. Other studies have suggested an action of genistein, independent of estrogen receptor, in inhibiting Ca2+ entry. Genistein reportedly (9) antagonized Ca2+ entry in isolated rabbit coronary artery rings and inhibited L-type Ca2+ currents in guinea pig ventricular myocytes through an action not affected by ICI. Genistein was reported to inhibit L-type Ca2+ channel currents in rat ventricular myocytes by acting as a tyrosine kinase inhibitor. The possible mechanism was reported (18) to be influencing the phosphorylation level and thus decreasing the open probability of the channel.

To our knowledge, this study is the first indication that dietary phytoestrogens have a protective role during global, hypothermic, cardioplegia-protected ischemia, followed by normothermic reperfusion in rats. Our experimental results suggest that the protective effects of dietary phytoestrogens are the following: 1) maintaining NO release through classical estrogen receptor-mediated mechanisms, 2) preserving mitochondrial structure and function through the function of classical estrogen receptor, and 3) attenuating myocardial Ca2+ accumulation through mechanisms independent of classical estrogen receptor.


    ACKNOWLEDGEMENTS

We greatly appreciate the assistance of Betty Ujhelyi, Joan Thompson, Sharon Meachum, Sherrie Lanzo, Alexander Reviera, and Dr. Victor Krylov. ICI-182,780 was a gift from Cancer and Infectious Research, Zeneca (Cheshire, United Kingdom). The soy protein and soy protein extract were gifts from Protein Technologies International (St. Louis, MO).


    FOOTNOTES

This work was supported by Illinois Council for Food and Agricultural Research Grant 99I-066-4.

Address for reprint requests and other correspondence: D. R. Gross, Dept. of Veterinary Biosciences, 3516 VMBS Bldg., 2001 S. Lincoln Ave., Urbana, IL 61802 (E-mail: dgross{at}cvm.uiuc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 May 2001; accepted in final form 21 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alkayed, NJ, Murphy SJ, Traystman RJ, and Hurn PD. Neuroprotective effects of female gonadal steroids in reproductively senescent female rats. Stroke 31: 161-168, 2000[Abstract/Free Full Text].

2.   Bourdillon, PD, and Poole-Wilson PA. The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res 50: 360-368, 1982[Abstract/Free Full Text].

3.   Coldham, NG, and Sauer MJ. Pharmacokinetics of [(14)C] genistein in the rat: gender-related differences, potential mechanisms of biological action, and implications for human health. Toxicol Appl Pharmacol 164: 206-215, 2000[ISI][Medline].

4.   Coward, L, Kirk M, Albin N, and Barnes S. Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin Chim Acta 247: 121-142, 1996[ISI][Medline].

5.   Davis, SR, Murkies AL, and Wilcox G. Phytoestrogens in clinical practice. Integr Med 1: 27-34, 1998.

6.   Deodato, B, Altavilla D, Squadrito G, Campo GM, Arlotta M, Minutoli L, Saitta A, Cucinotta D, Calapai G, Caputi AP, Miano M, and Squadrito F. Cardioprotection by the phytoestrogen genistein in experimental myocardial ischaemia-reperfusion injury. Br J Pharmacol 128: 1683-1690, 1999[ISI][Medline].

7.   Dubey, RK, Gillespie DG, Imthurn B, Rosselli M, Jackson EK, and Keller PJ. Phytoestrogens inhibit growth and MAP kinase activity in human aortic smooth muscle cells. Hypertension 33: 177-182, 1999[Abstract/Free Full Text].

8.   Engelman, DT, Watanabe M, Engelman RM, Rousou JA, Flack JE, III, Deaton DN, and Das DK. Constitutive nitric oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 110: 1047-1053, 1995[Abstract/Free Full Text].

9.   Figtree, GA, Griffiths H, Lu YQ, Webb CM, MacLeod K, and Collins P. Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. J Am Coll Cardiol 35: 1977-1985, 2000[Abstract/Free Full Text].

10.   Giraldez, RR, Panda A, Xia Y, Sanders SP, and Zweier JL. Decreased nitric-oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart. J Biol Chem 272: 21420-21426, 1997[Abstract/Free Full Text].

11.   Grinwald, PM. Calcium uptake during post-ischemic reperfusion in the isolated rat heart: Influence of extracellular sodium. J Mol Cell Cardiol 14: 359-365, 1982[ISI][Medline].

12.   Grohe, C, Kahlert S, Lobbert K, and Vetter H. Expression of oestrogen receptor alpha  and beta  in rat heart: role of local oestrogen synthesis. J Endocrinol 156: R1-R7, 1997.

13.   Guevara, I, Iwanejko J, Dembinxka-Kiee A, Bartus S, Malczewska-Malee M, and Szezudlik A. Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 274: 177-188, 1998[ISI][Medline].

14.   Honore, EK, Williams JK, Anthony MS, and Clarkson TB. Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques. Fertil Steril 67: 148-154, 1997[ISI][Medline].

15.   Itkonen, P, and Collan Y. Mitochondrial flocculent densities in ischemia, digestion experiments. Acta Pathol Microbiol Immunol Scand 91: 463-468, 1983.

16.   Jennings, RB, Herdson PB, and Sommers HM. Structural and functional abnormalities in mitochondria isolated from ischemic dog myocardium. Lab Invest 20: 548-557, 1969[ISI][Medline].

17.   Jovanovic, S, Jovanovic A, Shen WK, and Terzic A. Protective effects of 17 beta-estradiol in cardiac cells: implications for hyperkalemic cardioplegia. Ann Thorac Surg 66: 1658-1661, 1998[Abstract/Free Full Text].

18.   Katsube, Y, Yokoshiki H, Nguyen L, Yamamoto M, and Sperelakis N. Inhibition of Ca2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein. Eur J Pharmacol 345: 309-314, 1998[ISI][Medline].

19.   Kloner, RA, Ganote CE, Whaler D, and Jennings RB. Effect of a transient period of ischemia on myocardiac cells: II. Fine structure during the first few minutes of reflow. Am J Pathol 74: 399-442, 1974[ISI][Medline].

20.   Kuipper, G, Lemmen JG, Carlson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, and Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139: 4252-4263, 1998[Abstract/Free Full Text].

21.   Liu, Y, Peterson DA, Kimura H, and Schubert D. Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 69: 581-593, 1997[ISI][Medline].

22.   Lundh, TJO, Petersson H, and Kiesslilng KH. Liquid chromatographic determination of the estrogens daidzein, formononetic, coumestrol, and equol in bovine blood plasma and urine. J Assoc Anal Chem 71: 938-941, 1988.

23.   Makela, S, Savolainen H, Aavik E, Myllarniemi M, Strauss L, Taskinen E, Gustafsson JA, and Hayry P. Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors alpha and beta. Proc Natl Acad Sci USA 96: 7077-7082, 1999[Abstract/Free Full Text].

24.   Mattson, MP, Robinson N, and Guo Q. Estrogens stabilize mitochondrial function and protect against the pro-apoptotic action of mutant presenilin-1. Neuroreport 8: 3817-3821, 1997[ISI][Medline].

25.   Miksicek, RJ. Commonly occurring plant flavonoids have estrogen activity. Mol Pharmacol 44: 37-43, 1993[Abstract].

26.  Oliveira CA, Carnes K, Franca LR, and Hess RA. Infertility and testicular atrophy in the antiestrogen-treated adult male rat. Biol Repro. In press.

27.   Pabla, R, and Curtis MJ. Effect of endogenous nitric oxide on cardiac systolic and diastolic function during ischemia and reperfusion in the rat isolated perfused heart. J Mol Cell Cardiol 28: 2111-2121, 1996[ISI][Medline].

28.   Pappetropoulos, A, Rudic RD, and Sessa WC. Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res 43: 509-520, 1999[Abstract/Free Full Text].

29.   Pernow, J, and Wang QD. The role of L-arginine/nitric oxide pathway in myocardial ischaemic and reperfusion injury. Acta Physiol Scand 167: 151-159, 1999[ISI][Medline].

30.   Register, TC, and Adams AR. Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor beta. J Steroid Biochem Mol Biol 64: 187-191, 1998[ISI][Medline].

31.   Reibel, DK, and Rovetto MJ. Myocardial adenosine salvage rates and restoration of ATP content following ischemia. Am J Physiol Heart Circ Physiol 237: H247-H252, 1979.

32.   Rouslin, W. Identification of mitochondrial dysfunction at coupling site I: loss of activity of NADH-ubiquinone oxidoreductase during myocardial ischemia. In: Methods in Toxicology, Volume 2: Mitochondrial Dysfunction, edited by Lash LH, and Jones DP.. San Diego, CA: Academic, 1993, p. 311-323.

33.   Schaper, J, Heherlein F, Schlepper M, and Thiedemann KU. Ultrastructural alterations during ischemia and reperfusion in human hearts during cardiac surgery. J Mol Cell Cardiol 9: 175-189, 1977[ISI][Medline].

34.   Squadrito, F, Altavilla D, Squadrito G, Saitta A, Cucinotta D, Minutoli L, Deodato B, Ferlito M, Campo GM, Bova A, and Caputi AP. Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats. Cardiovasc Res 45: 454-462, 2000[Abstract/Free Full Text].

35.   Steenbergen, C, Murphy E, Levy L, and London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 60: 700-707, 1987[Abstract/Free Full Text].

36.   Wang, M, Yamamoto S, Chung HM, Chung SY, Miyatani S, Mori M, Okita T, and Sugano M. Antihypercholesterolemic effect of undigested fraction of soybean protein in young female volunteers. J Nutr Sci Vitaminol (Tokyo) 41: 187-195, 1995[Medline].

37.   Zhai, P, Eurell TE, Cooke PS, Lubahn DB, and Gross DR. Myocardial ischemia-reperfusion injury in estrogen receptor-alpha knockout and wild-type mice. Am J Physiol Heart Circ Physiol 278: H1640-H1647, 2000[Abstract/Free Full Text].

38.   Zhai, P, Eurell TE, Cotthaus R, Jeffery EH, Bahr JM, and Gross DR. Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats. Am J Physiol Heart Circ Physiol 279: H2766-H2775, 2000[Abstract/Free Full Text].

39.   Zheng, J, and Ramirez VD. Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitive-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase. J Steroid Biochem Mol Biol 68: 65-75, 1999[ISI][Medline].

Am J Physiol Heart Circ Physiol 281(3):H1223-H1232
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. J. Merkel, L. Liu, Z. Cao, W. Packwood, P. D. Hurn, and D. M. Van Winkle
Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes
Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H409 - H415.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. D. Gardner, G. L. Brower, T. G. Voloshenyuk, and J. S. Janicki
Cardioprotection in female rats subjected to chronic volume overload: synergistic interaction of estrogen and phytoestrogens
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H198 - H204.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Cao, L. Liu, W. Packwood, M. Merkel, P. D. Hurn, and D. M. Van Winkle
Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H302 - H310.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
N. Shimozawa, K. Okajima, and N. Harada
Estrogen and isoflavone attenuate stress-induced gastric mucosal injury by inhibiting decreases in gastric tissue levels of CGRP in ovariectomized rats
Am J Physiol Gastrointest Liver Physiol, February 1, 2007; 292(2): G615 - G619.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. D. Gardner, G. L. Brower, and J. S. Janicki
Effects of dietary phytoestrogens on cardiac remodeling secondary to chronic volume overload in female rats
J Appl Physiol, October 1, 2005; 99(4): 1378 - 1383.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
R. R. Magness, T. M. Phernetton, T. C. Gibson, and D.-b. Chen
Uterine blood flow responses to ICI 182 780 in ovariectomized oestradiol-17{beta}-treated, intact follicular and pregnant sheep
J. Physiol., May 15, 2005; 565(1): 71 - 83.
[Abstract] [Full Text] [PDF]

Home page
 Integr Cancer TherHome page
R M Fleming
What Effect, if any, does Soy Protein have on Breast Tissue?
Integr Cancer Ther, September 1, 2003; 2(3): 225 - 228.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (16)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhai, P.
Right arrow Articles by Gross, D. R.